Cell Surface Glycolipids - American Chemical Society

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Preparative and Analytical High Performance Liquid Chromatography of Glycolipids R. H. MC CLUER Eunice Kennedy Shriver Center, Waltham, MA 02154 M. D. ULLMAN Center for Disease Control, Atlanta, GA 30333 High performance liquid chromatography (HPLC) implies the use of reusable columns, injection port sample application, the use of pumps for uniform solvent flow operated at high pressures if necessary and automatic on-line sample detection. The availability of a large variety of microparticulate column packing materials, efficient column packing techniques, high pressure-low volume pumping equipment and various types of highly sensitive detectors have led to the development of sensitive, rapid and quantitative methods, analogous to that available for volatile materials by gas-chromatography, for the isolation and analysis of a large variety of relatively high molecular weight substances of biological interest (1). We have attempted to utilize these tools of modern liquid chromatography to develop rapid and highly sensitive methods for the analysis of glycolipids. Our early experience with HPLC techniques indicated that the analysis of glycolipids becomes interestingly sensitive and practical if derivatives are prepared that allow the use of ultraviolet detectors and that exhibit good chromatographic properties. We have primarily studied the preparation of the benzoyl derivatives of glycolipids for the development of analytical and preparative HPLC methods. The following is a concise review of studies with neutral glycosphingolipids with emphasis on recent work in which we have utilized p-dimethylaminopyridine (DMAP) as a catalyst to effect benzoylation with benzoic anhydride. Preparation and analysis of benzoylated cerebrosides We initially demonstrated that brain cerebrosides, galactosylceramides containing hydroxy fatty acids (HFA) and nonhydroxy fatty acids (NFA), could be completely derivatized by reaction with 10% benzoyl chloride at 60OC for 1 hour (2). After removal of excess reagents by partition between hexane and alkaline aqueous methanol, the perbenzoyl derivatives were seen to separate into two completely resolved components (HFA and NFA 0-8412-0556-6/ 80/ 47-128-001 $5.00/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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C E L L SURFACE GLYCOLIPIDS

c e r e b r o s i d e s ) by adsorption chromatography on a p e l l i c u l a r s i l i c a column support ( Z i p a x , E . I . DuPont) w i t h methanol i n pentane as the e l u t i n g s o l v e n t and 280 nm d e t e c t i o n . Hexane-ethyl acetate was subsequently shown to be a s u p e r i o r e l u t i n g solvent because regeneration of the column adsorbant a c t i v i t y was more r e p r o ducible (3). Attempts to recover the parent cerebrosides by treatment of the benzoyl d e r i v a t i v e s w i t h m i l d a l k a l i was successful w i t h the HFA-cerebrosides, but the NFA-cerebroside d e r i v a t i v e gave r i s e to benzoyl psychosine as well as the parent NFA-cerebroside. This was demonstrated to r e s u l t from the d i a c y l amine s t r u c t u r e of the perbenzoyl NFA-cerebroside. NMR s t u d i e s of the two cerebroside d e r i v a t i v e s i n d i c a t e d the presence of s i x benzoyl groups i n each case and the presence of an amide proton i n the H F A - d e r i v a t i v e which was absent i n the NFA-cerebroside d e r i v a t i v e . As reported by Inch and F l e t c h e r (4) f o r the diacylamine d e r i v a t i v e s of amino s u g a r s , the N-acyl groups are randomly removed during a l k a l i h y d r o l y s i s thus cerebrosides and other s p h i n g o l i p i d s which contain non-hydrox:y f a t t y acids or N a c e t y l amino sugars cannot be recovered i n high y i e l d s because a l k a l i n e h y d r o l y s i s of the perbenzoyl d e r i v a t i v e r e s u l t s i n the formation of N-benzoyl compounds as well as the parent N-acyl s p h i n g o l i p i d . Benzoylation of cerebrosides w i t h 10% benzoic anhydride i n p y r i d i n e was shown to lead only to the formation of 0 - a c y l d e r i v a t i v e s and the parent g l y c o l i p i d s could be recovered a f t e r a l k a l i n e methanolysis; however, t h i s r e a c t i o n was s l u g g i s h and r e q u i r e d treatment a t 110 C f o r 18 hours f o r completion. S u l f a t i d e s were shown to be completely converted to benzoylated cerebrosides during t h i s anhydride r e a c t i o n . We chose the benzoyl c h l o r i d e r e a c t i o n f o r a n a l y t i c a l purposes because r e a c t i o n times were shorter and s u l f a t i d e s do not d e s u l f a t e under c o n d i t i o n s r e q u i r e d f o r cerebroside d e r i v a t i z a t i o n . Because s p h i n g o l i p i d s which c o n t a i n only hydroxy f a t t y a c i d s as N-acyl s u b s t i t u e n t s form the same d e r i v a t i v e with e i t h e r the benzoyl c h l o r i d e or the anhydride r e a c t i o n , they can be e a s i l y d i s t i n g u i s h e d from nonhydroxy f a t t y a c i d c o n t a i n i n g s p h i n g o l i p i d s which form d i f f e r e n t d e r i v a t i v e s , d i s t i n q u i s h a b l e by HPLC, when benzoylated with the c h l o r i d e as compared to the anhydride r e a c t i o n . A n a l y s i s of

ceramides

A q u a n t i t a t i v e HPLC method f o r the a n a l y s i s of s p h i n g o l i p i d s as t h e i r perbenzoyl d e r i v a t i v e s was f i r s t developed f o r ceramides (5). Ceramides can be conveniently d e r i v a t i z e d w i t h benzoic anhydride i n p y r i d i n e (3 hrs at 110°C) and the products formed have been u t i l i z e d f o r the q u a n t i t a t i v e a n a l y s i s of NFA and HFA ceramides i n normal and F a r b e r ' s disease t i s s u e . Iwamori and Moser a l s o u t i l i z e d t h i s procedure f o r the a n a l y s i s of ceramides i n F a r b e r ' s disease u r i n e ( 6 ) . More r e c e n t l y Iwamori and Moser (7) e s t a b l i s h e d that the ceramide d e r i v a t i v e s formed by r e a c t i o n with benzoyl c h l o r i d e or benzoic anhydride are analogous to those formed with c e r e b r o s i d e s . They a l s o c h a r a c t e r i z e d the behavior

Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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of ceramides t h a t contain phytosphingosine and described the use of estrone as an i n t e r n a l s t a n d a r d . These published ceramide methods u t i l i z e d 280 nm d e t e c t i o n and the s e n s i t i v i t y of the procedures could e a s i l y be increased to the pmole l e v e l by d e t e c t i o n a t 230 nm i f a v a r i a b l e wave length d e t e c t o r i s u t i l i z e d . A l s o , the speed of d e r i v a t i z a t i o n could undoubtedly be g r e a t l y increased by the use of the DMAP as a c a t a l y s t as described below f o r neutral g l y c o s p h i n g o l i p i d s . Samuelsson (8) u t i l i z e d elegent gas chromatograph-mass spectrometric (GC-MS) methods f o r the a n a l y s i s of ceramide molecular s p e c i e s , but HPLC methods o f f e r the advantages of n o n - d e s t r u c t i v e measurement so that components can e a s i l y be c o l l e c t e d f o r determination of r a d i o a c t i v i t y or f o r f u r t h e r a n a l y s i s . Q u a n t i t a t i v e a n a l y s i s of n e u t r a l g l y c o s p h i n g o l i p i d s An HPLC method f o r neutral g l y c o s p h i n g o l i p i d s was f i r s t designed f o r the a n a l y s i s of human plasma g l y c o l i p i d s ( 3 ) , which c o n s i s t p r i m a r i l y of glucosylceramide, l a c t o s y l c e r a m i d e , g l o b o t r i a o s y l c e r a m i d e and globotetraosylceramide ( g l o b o s i d e ) . Conditions f o r the simultaneous d e r i v a t i z a t i o n of t h i s group of compounds, which provided maximal y i e l d s f o r g l o b o s i d e , were s e l e c t e d to be 37 C f o r 16 hours i n 10% benzoyl c h l o r i d e i n p y r i d i n e , s l i g h t l y d i f f e r e n t from those p r e v i o u s l y u t i l i z e d f o r cerebrosides. S a t i s f a c t o r y chromatographic c o n d i t i o n s , which provided base l i n e r e s o l u t i o n of these four d e r i v a t i v e s i n a minimum of t i m e , were found w i t h the Zipax column and a gradient of e t h y l acetate i n hexane and 280 nm d e t e c t i o n . With t h i s chromatographic system the standard g l y c o l i p i d d e r i v a t i v e s could be separated and q u a n t i t a t e d i n l e s s than 20 min and column a c t i v i t y could be r e p r o d u c i b l y regenerated i n e i g h t min.. Less than 20 nmole of each g l y c o l i p i d could be e a s i l y q u a n t i t a t e d w i t h t h i s procedure. The u t i l i t y of ethyl acetate i n t h i s chromatographic system was e x c e l l e n t but prevented the use o f d e t e c t i o n below 260 nm. Because the max of the benzoyl d e r i v a t i v e s i s a t 230nm we sought chromatographic solvents which could be u t i l i z e d a t t h i s wavel e n g t h , s t i l l provide adequate chromatographic r e s o l u t i o n and a l l o w r a p i d l y r e - e q u i l i b r a t i o n of column adsorbant a c t i v i t y a f t e r gradient e l u t i o n . A dioxane-hexane s o l v e n t system proved adequate except r e s i d u a l l i g h t absorption due to the dioxane produced an undesirable r i s i n g base l i n e during the g r a d i e n t . The r i s i n g base l i n e was e l i m i n a t e d by d i r e c t i n g the s o l v e n t flow through a precolumn p r e - i n j e c t o r high pressure reference c e l l . This path generates a h o r i z o n t a l b a s e l i n e with a negative and p o s i t i v e d e f l e c t i o n a t the beginning and end of the g r a d i e n t r e s p e c t i v e l y . With t h i s system r e l i a b l e q u a n t i t a t i o n of l e s s than 50 pmoles of each of the four major plasma g l y c o s p h i n g o l i p i d s can be obtained (9). For the a n a l y s i s of plasma g l y c o l i p i d s i t i s necessary to f i r s t i s o l a t e a g l y c o l i p i d f r a c t i o n by s o l v e n t e x t r a c t i o n , b

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chromatography on small U n i s i l columns and treatment w i t h m i l d a l k a l i as o r i g i n a l l y described by Vance and Sweeley (10). C o n s i s t e n t r e c o v e r i e s of the g l y c o l i p i d s i s dependent upon maintaining a f i x e d r a t i o between sample s i z e and the q u a n t i t y of U n i s i l employed. Accuracy o f the method i s improved by the u t i l i z a t i o n of an i n t e r n a l standard such as N-acetylpsychosine which i s added to the plasma samples p r i o r to the i n i t i a l l i p i d e x t r a c t i o n . One ml plasma samples are now r o u t i n e l y used f o r g l y c o l i p i d a n a l y s i s although s e n s i t i v i t y of the HPLC procedure t h e o r e t i c a l l y should a l l o w a n a l y s i s o f l e s s than 0.1 m l . However, the i s o l a t i o n o f such small q u a n t i t i e s of g l y c o l i p i d p r i o r to d e r i v a t i z a t i o n present d i f f i c u l t recovery problems. The high s e n s i t i v i t y of the d e t e c t i o n system employed f o r the a n a l y s i s of pmole q u a n t i t i e s a l s o r e q u i r e s precautions so t h a t UV absorbing contaminates are not introduced during processing of the samples. A l l glassware should be scrupulously c l e a n and HPLC grade s o l v e n t s should be used f o r a l l steps i n the i s o l a t i o n and chromatography. This HPLC procedure has a l s o been u t i l i z e d f o r the a n a l y s i s of neutral g l y c o s p h i n g o l i p i d s from a v a r i e t y o f sources. Human e r y t h r o c y t e s , p e r i p h e r a l leukocytes and l i v e r have been s a t i s f a c t o r i l y a n a l y z e d , but i t should be recognized t h a t each d i f f e r e n t t i s s u e source may r e q u i r e d i f f e r e n t e x t r a c t i o n c o n d i t i o n s and modified s o l v e n t g r a d i e n t e l u t i o n i n order to o b t a i n maximal r e c o v e r i e s and optimal chromatographic r e s o l u t i o n of the t i s s u e c h a r a c t e r i s t i c g l y c o s p h i n g o l i p i d s . F l e t c h e r , Bremer and Schwarting (11) have optimized the procedure f o r the a n a l y s i s of e r y t h r o c y t e g l y c o l i p i d s and demonstrated t h a t e r y t h r o c y t e s from blood group P-| i n d i v i d u a l s c o n t a i n more g l o b o t r i a o s y l c e r a m i d e and l e s s l a c t o s y l c e r a m i d e than e r y t h r o c y t e s from blood group ?2 i n d i v i d u a l s . The dramatic sex d i f f e r e n c e i n mouse kidney g l y c o l i p i d s and the occurrence of l a r g e amounts of g l y c o l i p i d s i n male mouse u r i n e was r e a d i l y demonstrated by these HPLC methods. The l i g h t ear ( l e / l e ) mouse pigmentation mutant was shown to have storage of g l y c o l i p i d s i n t h e i r kidneys which i s apparently due to an abnormality i n the s e c r e t i o n of m u l t i l a m e l l a r lysosomal bodies t h a t c o n t a i n l a r g e amounts of g l y c o s p h i n g o l i p i d s (12) . Thus, the a n a l y t i c a l HPLC method f o r g l y c o l i p i d s i s proving useful f o r a v a r i e t y o f s t u d i e s r e l a t e d to g l y c o s p h i n g o l i p i d f u n c t i o n and metabolism. Other useful a n a l y t i c a l HPLC procedures f o r the a n a l y s i s of d e r i v a t i z e d g l y c o l i p i d s have been developed. Nanaka and Kishimoto (13) have devised an HPLC procedure which allows the t i s s u e l e v e l s of NFA c e r e b r o s i d e , HFA c e r e b r o s i d e , NFA s u l f a t i d e , HFA s u l f a t i d e , and monogalactosyl d i g l y c e r i d e to be determined s i m u l t a n e o u s l y . This procedure i n v o l v e s benzoyl a t i o n of t o t a l l i p i d e x t r a c t s , d e s u l f a t i o n w i t h m i l d a c i d and subsequent chromatography w i t h the r a d i e n t of isopropanol i n hexane. S u s u k i , Honda and Yamakawa 14) prepared a c e t y l a t e d g l y c o l i p i d which were subsequently reacted w i t h p - n i t r o b e n z o y l c h l o r i d e to form the O - a c e t y l - N - p nitrobenzoyl d e r i v a t i v e s which have good chromatographic


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p r o p e r t i e s and can be detected w i t h high s e n s i t i v i t y w i t h a s i n g l e wavelength detector a t 254 nm. While o f f e r i n g these advantages, t h i s procedure cannot be u t i l i z e d w i t h g l y c o l i p i d s t h a t c o n t a i n only a-hydroxy f a t t y a c i d s and no amino sugar. A l l of the benzoylated or O - a c e t y l - N - p - n i t r o b e n z o y l d e r i v a t i v e s can be u s e f u l l y separated i n t o molecular species by reverse phase chromatography ( 1 1 , 1 2 ) . We have r e c e n t l y shown t h a t the use o f the c a t a l y s t N - d i methylaminopyridine (DMAP) w i t h benzoic anhydride g r e a t l y a c c e l e r a t e s the d e r i v a t i z a t i o n w i t h t h i s reagent (13). Reaction w i t h DMAP and the anhydride avoids amide a c y l a t i o n , forms s i n g l e products w i t h s a t i s f a c t o r y chromatographic p r o p e r t i e s and parent g l y c o s p h i n g o l i p i d s can be regenerated by m i l d a l k a l i n e h y d r o l y s i s . For a n a l y t i c a l purposes, t h i s r e a c t i o n has been u t i l i z e d f o r the a n a l y s i s of plasma n e u t r a l g l y c o s p h i n g o l i p i d s . The g l y c o l i p i d s were reacted w i t h 20% benzoic a c i d a n h y d r i d e , 5% DMAP i n p y r i d i n e at 37°C f o r four hours. G l c C e r , LacCer, Gb0se Cer and Gb0se Cer each gave s i n g l e r e a c t i o n products w i t h maximum y i e l d s w i t h r e a c t i o n times between 2 and 6 hours. Excess reagents were removed from the products by p a r t i t i o n between hexane and aqueous a l k a l i n e methanol as described p r e v i o u s l y f o r the benzoyl c h l o r i d e products. The products were than analyzed w i t h the Zipax column and dioxane g r a d i e n t a l s o as p r e v i o u s l y described ( 3 ) . The chromatographic a n a l y s i s o f the per-O-benzoylated glycosphingol i p i d standards and plasma g l y c o s p h i n g o l i p i d s are shown i n F i g . 1 along w i t h the e l u t i o n p a t t e r n of plasma g l y c o l i p i d d e r i v a t i v e s obtained by r e a c t i o n w i t h benzoyl c h l o r i d e . The d e r i v a t i v e s obtained by r e a c t i o n with benzoic anhydride have longer r e t e n t i o n times when compared to the benzoyl c h l o r i d e products. We have p r e v i o u s l y shown t h a t galactosylceramide which contains a-hydroxy f a t t y a c i d s i s not N-benzoylated w i t h benzoyl c h l o r i d e and r e a c t i o n w i t h benzoic anhydride or benzoyl c h l o r i d e r e s u l t s i n an i d e n t i c a l product. S i m i l a r r e s u l t s have been obtained w i t h anhydride i n the presence of DMAP as i l l u s t r a t e d i n F i g . 2. The behavior of peak b" which we have shown to be derived from a-hydroxy f a t t y a c i d c o n t a i n i n g g l u c o s y l and galactosylceramides i s i l l u s t r a t i v e . The UV response from each of the standard GSLs benzoylated by the anhydride and by the benzoyl c h l o r i d e method were compared. The r e l a t i v e responses ( c h l o r i d e / a n h y d r i d e ) f o r the mono, d i , t r i and t e t r a - h e x o s y l ceramide were found to be 1.18, 1.15, 0.94 and 1.03 r e s p e c t i v e l y . These values were not s i g n i f i c a n t l y d i f f e r e n t from c a l c u l a t e d r a t i o n s 1.20, 1.12, 1.09, and 1.15, based on the assumption t h a t the anhydride method avoids amide b e n z o y l a t i o n . The y i e l d s of the per-O-benzoylated products were s i m i l a r to those obtained f o r the products o f the benzoyl c h l o r i d e method reported p r e v i o u s l y . The parent GSLs can be regenerated from t h e i r p e r - O benzoylated products by treatment w i t h m i l d a l k a l i . Globoside was benzoylated by both methods, the products subjected to HPLC, and the peaks c o l l e c t e d and t r e a t e d w i t h 0.5N methanolic sodium 3

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TIME (min) Figure 1.

HPLC of benzoylated standard and plasma glycosphingolipids

The derivatized glycosphingolipids were injected onto a Zipex column (2.1 mm X 50 cm) and eluted with a 13-min linear gradient of 2.5-25% dioxane in hexane with detection at 230 nm. A. Standard glycosphingolipids (GSL) per-O-benzoylated with benzoic anhydride and 4-dimethylaminopyridine (DMAP). B. Plasma GSL per-O-benzoylated with benzoic anhydride and DMAP. C. Plasma GSL perbenzoylated with benzoyl chloride. Glycosphingolipid peaks are identified as: (1) glycosylceramide, (2) lactosylceramide, (3) galactosyl-lactosylceramide, (4) N-acetylgalactosaminylgalactosyllactosylceramide. Peak A is unidentified, and peak B is hydroxy fatty acid containing galactosylceramide.


Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980. Preparative isolation of liver glycolipids

A glycosphingolipid fraction (15 mg) was benzoylated with DMAP and benzoic anhydrydride, and the derivatives were chromatographed on a LiChrosorb SI 100 column with an ethyl acetate in hexane gradient as described in the text. Detection was at 280 nm. Components eluting at 7, 9, and 49 min are unidentified.

Figure 2.

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hydroxide f o r 1 hour a t 37°C. A f t e r s o l v e n t p a r t i t i o n , i n C/M/H 0 ( 8 / 3 / 3 ) the lower phase l i p i d products were examined by TLC and v i s u a l i z e d under UV l i g h t and w i t h o r c i n o l and s u l f u r i c a c i d spray reagents. Only a s i n g l e product, with no UV absorption was obtained from the anhydride benzoylated g l o b o s i d e . Methanolysis and GLC a n a l y s i s of f a t t y a c i d s from t h i s product revealed t h a t t h e i r composition was unchanged compared to t h a t of the parent globoside. The r a t i o of 24:1 to 24:0 f a t t y a c i d s was 0.51 i n the o r i g i n a l sample and 0.50 i n the debenzoylated sample. The use o f benzoic anhydride w i t h DMAP as a c a t a l y s t provides a convenient means f o r the p r e p a r a t i o n o f the p e r - O benzoylated d e r i v a t i v e s of GSLs. These d e r i v a t i v e s can subsequently be u t i l i z e d f o r a n a l y t i c a l and p r e p a r a t i v e HPLC because parent GSLs can be c o n v e n i e n t l y recovered i n high y i e l d s by m i l d a l k a l i n e h y d r o l y s i s . 2

P r e p a r a t i v e HPLC of per-O-benzoylated g l y c o s p h i n g o l i p i d s . We d e s c r i b e here a procedure f o r the convenient d e r i v a t i z a t i o n and p r e p a r a t i v e i s o l a t i o n o f g l y c o l i p i d s by HPLC w i t h UV d e t e c t i o n a t 280 nm. Previous t h i n - l a y e r chromatography (TLC), l i q u i d chromatography (LC) and HPLC procedures have been encumbered by the l a c k of a convenient n o n - d e s t r u c t i v e method of d e t e c t i o n f o r the components o f i n t e r e s t . F u r t h e r , TLC i s o l a t i o n s are hampered by the small load c a p a c i t i e s of each p l a t e which r e q u i r e s the s t r e a k i n g , scraping and e l u t i o n of compounds from m u l t i p l e p l a t e s and by the a m b i g u i t i e s introduced by l i g h t l y spraying of each p l a t e w i t h a n o n - d e s t r u c t i v e spray such as methanol-water 1:1 ( v / v ) or p r i m u l i n e (15). T h i c k - l a y e r TLC, which allows l a r g e l o a d s , f r e q u e n t l y y i e l d s poor r e s o l u t i o n because the streaked sample tends to " f l a r e " as i t penetrates the separation bed, thus causing s i g n i f i c a n t overlap of bands d u r i n g m i g r a t i o n . Larger q u a n t i t i e s of g l y c o l i p i d s have been separated by L C , w i t h v a r y i n g degrees o f success, on such column packing m a t e r i a l s as alumina ( 1 6 , 1 7 ) , A n a s i l S ( 1 8 , 1 9 , 2 0 ) , F l o r i s i l ( 2 1 , 2 2 , 2 3 , 2 4 , 2 5 ) , Iatrobeads (26), s i l i c i c a c i d (27,28,29) S i l i c a gel G ( 3 0 ) , and U n i s i l ( 3 1 , 3 2 ) . A l l of these LC procedures are hampered by the absence of an adequate d e t e c t i o n system. Although the l a c k of o n - l i n e d e t e c t i o n has impeded the adaptation of the LC procedures to HPLC, p r e p a r a t i v e HPLC of g l y c o l i p i d s has been performed on s i l i c a SI 60 w i t h post-column, o f f - l i n e TLC d e t e c t i o n (30) and w i t h a moving w i r e detector ( 3 1 ) . The procedure described below f o r p e r - 0 - b e n z o y l a t i o n of g l y c o l i p i d s w i t h benzoic anhydride i n p y r i d i n e and DMAP as c a t a l y s t avoids N-benzoylation problem and provides a convenient method f o r the d e t e c t i o n and p r e p a r a t i v e i s o l a t i o n of g l y c o l i p i d s . The a p p l i c a t i o n of t h i s procedure f o r the i s o l a t i o n of 15 mg of g l y c o l i p i d s i n a s i n g l e HPLC run i s d e s c r i b e d . a . I s o l a t i o n of crude l i v e r n e u t r a l g l y c o l i p i d s . Total l i p i d s were i s o l a t e d from human l i v e r by the method of Folch e t a l . (33).


1.

MCCLUER AND ULLMAN

Liquid

Chromatography

9

Crude n e u t r a l g l y c o l i p i d s were i s o l a t e d from the l i p i d e x t r a c t e s s e n t i a l l y by the procedure of Vance and Sweeley (10). The l i p i d s were d i s s o l v e d i n chloroform and placed on a s i l i c i c a c i d column (25 gm packing to 1 gm l i p i d e x t r a c t ) the n e u t r a l l i p i d s and f a t t y a c i d s were e l u t e d w i t h chloroform (20 ml/gm p a c k i n g ) . Crude n e u t r a l g l y c o l i p i d s were then e l u t e d from the column w i t h acetone-methanol (9:1 v / v ) (40 ml/gm p a c k i n g ) . The acetonemethanol e l u a t e was c o l l e c t e d i n 200 ml f r a c t i o n s , so t h a t monoand d i - h e x o s y l ceramides were i n greater c o n c e n t r a t i o n i n the e a r l i e r f r a c t i o n s r e l a t i v e to the t r i - and t e t r a - h e x o s y l ceramides. The acetone-methanol from each of the d e s i r e d f r a c t i o n s was evaporated to dryness and exposed to m i l d a l k a l i n e m e t h a n o l y s i s . The content of n e u t r a l g l y c o l i p i d s i n the f r a c t i o n s of i n t e r e s t was determined by the q u a n t i t a t i v e HPLC method o f Ullman and McCluer ( 3 ) . b. P e r - Q - b e n z o y l a t i o n c o n d i t i o n . Samples which had been d r i e d under a stream of n i t r o g e n and which contained approximately 15 mg of n e u t r a l g l y c o l i p i d s from l i v e r , were t r a n s f e r r e d i n t o a 20 mm x 150 mm screw capped c u l t u r e tube and d e s s i c a t e d over P2O5 f o r a t l e a s t three hours. A 1.5 ml p o r t i o n of f r e s h l y prepared 20% benzoic anhydride i n p y r i d i n e (w/v) was added to the c u l t u r e tube followed by a 1.5 ml p o r t i o n of l $ DMAP i n p y r i d i n e (v/v).The tube was b r i e f l y flushed w i t h n i t r o g e n , capped t i g h t l y , and incubated at 37°C f o r two hours. The tube was then placed i n a water bath maintained a t room temperature and the p y r i d i n e was removed w i t h a stream of n i t r o g e n . Three ml of hexane was added to the r e s i d u e and the suspension was washed four times with 1.8 ml of a l k a l i n e methanol. The a l k a l i n e methanol was prepared by the a d d i t i o n of 1.2 gm Na2C03 to 300 ml of methanol-water 80:20 ( v / v ) ( a l l of the Na2C03 d i d not d i s s o l v e ) . Each time the lower phase was withdrawn and d i s c a r d e d . F i n a l l y , the hexane l a y e r was washed once w i t h 1.8 ml of methanol-water 80:20 and a f t e r removal of the lower phase the hexane was evaporated w i t h a stream of n i t r o g e n at room temperature. The sample was then d i s s o l v e d i n 5 ml of methanol and placed onto a reverse phase r a p i d sample p r e p a r a t i o n column (Sep PakTM,Waters A s s o c . ) t h a t had been preconditioned w i t h 30 ml of methanol. An a d d i t i o n a l wash of 5 ml of methanol was added to the column and the per-O-benzoylated g l y c o l i p i d s were e l u t e d w i t h 10 ml of methanol-acetone 9:1 i n a 20 mm x 150 mm screw cap c u l t u r e tube. This f r a c t i o n was d r i e d at room temperature w i t h a stream of n i t r o g e n and r e d i s s o l v e d i n 4% e t h y l acetate i n hexane ( v / v ) f o r i n j e c t i o n . c. HPLC. Per-O-benzoylated n e u t r a l g l y c o l i p i d s were a p p l i e d to a 4.6 mm x 25 cm LiChrosorb SI 100, 10y p a r t i c l e w i t h a loop i n j e c t o r and 4% e t h y l acetate i n hexane pumped a t 0.5 m l / m i n . The d e r i v a t i v e s were then e l u t e d i s o m a t i c a l l y w i t h 18% e t h y l acetate i n hexane f o r 30 minutes and then w i t h a l i n e a r g r a d i e n t of 18 to 45% e t h y l acetate i n hexane over t h i r t y minutes. The


10

CELL SURFACE GLYCOLIPIDS

flow r a t e was 3 ml/min and UV d e t e c t i o n was performed a t 280 nm. Each peak was i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d f o r recovery and p u r i t y by a n a l y t i c a l HPLC. A f t e r the i s o l a t i o n was complete the i n i t i a l s o l v e n t and adsorbant c o n d i t i o n s were regenerated w i t h a three minute reverse g r a d i e n t . d. R e s u l t s . E x c e l l e n t r e s o l u t i o n of the major g l y c o l i p i d peaks ( F i g . 2) was o b t a i n e d . Because of the mono-, d i - , t r i - , and t e t r a - h e x o s y l ceramides d i d not maintain a constant r a t i o i n each f r a c t i o n during the i s o l a t i o n of the crude g l y c o l i p i d f r a c t i o n from U n i s i l , the chromatogram i n Figure 2 i s not r e p r e s e n t a t i v e of the d i s t r i b u t i o n of l i v e r g l y c o l i p i d s . E t h y l a c e t a t e was s e l e c t e d as a s o l v e n t because of the high l i p i d s o l u b i l i t y of the d e r i v i t i z e d g l y c o l i p i d s because i t i s e a s i l y removed a f t e r c o l l e c t i o n of the d e s i r e d f r a c t i o n s , and because column adsorbant a c t i v i t y i s r a p d i l y reestablished a f t e r gradient e l u t i o n . Detection at 280nm avoided s a t u r a t i o n of the detector s i g n a l when l a r g e q u a n t i t i e s of g l y c o l i p i d s were a p p l i e d to the column. Larger columns and greater amounts o f g l y c o l i p i d s could have been used by s e t t i n g the d e t e c t o r to a s l i g h t l y higher wavelength thus decreasing the s e n s i t i v i t y . Although c a p a c i t y o f the column was not determined i n these experiments, there was no change i n r e t e n t i o n time or peak shape when 5, 10, or 15 mg of g l y c o l i p i d s were chromatographed. Each sample was c o l l e c t e d so t h a t approximately 5% of the l e a d i n g and t a i l i n g edges of the peaks were omitted from the c o l l e c t i o n . A f t e r a sample was c o l l e c t e d , the i s o l a t e d f r a c t i o n was d r i e d under n i t r o g e n , d i s s o l v e d i n a known volume of hexane and an a l i q u o t t e s t e d f o r p u r i t y by a n a l y t i c a l HPLC. The sample was then d r i e d and the residue was subjected to m i l d a l k a l i n e h y d r o l y s i s (to recover natural g l y c o l i p i d ) , perbenzoylated i n 10% benzoyl c h l o r i d e i n p y r i d i n e , r e d i s s o l v e d i n hexane and again analyzed by q u a n t i t a t i v e HPLC. The two methods f o r e v a l u a t i o n of f r a c t i o n p u r i t y were i n good agreement. U s u a l l y , i s o l a t e d sample peaks were greater than 98% free of g l y c o l i p i d contaminates and i t was not uncommon to i s o l a t e peaks t h a t contained no other glycolipids. I f necessary, f u r t h e r p u r i f i c a t i o n could be obtained by rerunning each of the i s o l a t e d samples i n an i s o c r a t i c s o l v e n t system with a s o l v e n t composition near the e l u t i n g composition of the g r a d i e n t . Each f r a c t i o n was shown to be free of non-UV absorbing i m p u r i t i e s by m i g r a t i o n of the i s o l a t e d natural g l y c o l i p i d s o n s i l i c a Gel G TLC p l a t e s i n chloroform-methanol water (65:25:4) w i t h d e t e c t i o n by c h a r r i n g w i t h 55% H2SO4 i n water (w/w) To determine the recovery of the neutral g l y c o l i p i d s a crude g l y c o l i p i d was per-O-benzoylated and 0.1% of the sample was i n j e c t e d onto the a n a l y t i c a l chromatograph to determine the amount of each component. The remainder was i n j e c t e d onto the p r e p a r a t i v e column and the t o t a l e f f l u e n t , e x c l u d i n g the s o l v e n t f r o n t was c o l l e c t e d , d r i e d under n i t r o g e n , d i s s o l v e d i n i n i t i a l s o l v e n t and


1. MCCLUER AND ULLMAN

Liquid Chromatography

11

0.1% reinjected onto the analytical column. Two different experiments averaged 84, 84.5, 86, and 89% recovery for mono-, di-, t r i - , and tetra- hexosyl ceramides, respectively. After completion of the preparative run, the column was regenerated and reused several times with little loss of efficiency. The use of the rapid sample preparation column in the isolation of the per-O-benzoylated derivatives was necessitated by the large quantity of dark brown impurities in the original lipid preparation, which also eluted from the Unisil column with the crude glycolipid fraction. The use of a Sep-Pak column greatly reduced this discoloration. The isocratic portion of the column elution was utilized to obtain better resolution of the other, early-eluting peaks which were assumed to be monohexosyl-containing sphingolipids. The structures of these components are understudy and presumably are similar to the "a" and "b" components seen with the plasma glycolipids in Fig. 1. Conclusions Derivatives of glycosphingolipids which have large extinction coefficients can be prepared and separated according to their carbohydrate content by adsorbtion chromatography. This use of modern HPLC equipment allows quantitation of less than 50 pmole quantities of these compounds so that small amounts of body fluids tissue or tissue culture cells can be readily analyzed for the major nuetral glycosphingolipid components. Such components can be further separated into molecular species by reverse phase chromatography. The use of DMAP as a catalyst for derivatization with benzoic acid anhydride allows the convenient preparation of per-0-benzoyl derivatives. Parent glycosphingolipids can be regenerated from these derivatives by treatment with mild alkali. Thus, modern liquid chromatographic techniques with on-line detection can be utilized for the isolation of the neutral glycosphingolipids.

m

Literature Cited 1. Snyder, L.R., and Kirkland, J . J . , "Introduction to Modern Liquid Chromatography"; Wiley-Interscience: New York, N.Y., 1974. 2. McCluer, R.H.; Evans, J.E. Preparation and analysis of benzoylated cerebrosides. J. Lipid Res., 1973, 14, 611. 3. Ullman, M.D., McCluer, R.H. Quantitative analysis of plasma neutral glycosphingolipids by high performance liquid chromatography of their perbenzoyl derivatives. J. Lipid Res., 1977, 18, 371-377. 4. Inch, T.D.; Fletcher, H.C. N-acyl derivatives of 2-acylamino2-deoxy-D-glucopyranose. J. Org. Chem., 1966, 31, 1815. 5. Sugita, M.; Iwamori, M; Evans, J.; McCluer, R.H.; Moser, H.W.; Dulaney, J.T. High-performance liquid chromatography of ceramides: application to analysis in human tissues and demonstration of ceramide excess in Farber's disease. J. Lipid Res., 1974, 15, 223.


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8. 9. 10. 11. 12. 13.

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15. 16. 17. 18. 19. 20.

21.

CELL SURFACE GLYCOLIPIDS

Iwamori, M.G.; Moser, H.W. Above normal urinary excretion of urinary ceramides in Farber's disease, and characterization of their components by high performance liquid chromatography. Clin. Chem., 1975, 21, 725. Iwamori, M.; Costello, C.; and Moser, H.W. Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography. J. Lipid Res., 1979, 20, 86. Samuelsson, K. Identification and quantitative determination of ceramides in human plasma. Scand. J. Clin. Lab. Invest., 1971, 27, 371. Ullman, M.D.; McCluer, R.H. Quantitative microanalysis of perbenzoylated glycosphingolipids by HPLC with detection at 230 nm. J. Lipid Res., 1978, 19, 910. Vance, D.E.; Sweely, C.C. Quantitative determination of the neutral glycosyl ceramides in human blood. J. Lipid Res., 1967, 8, 621-630. Fletcher, K.S.; Bremer, E.G.; Schwarting, G.A. P blood group regulation of glycosphingolipid levels in human erythrocytes. J. Biol. Chem., in press. McCluer, R.H.; Gross, S.K.; Sapirstein, V.S.; Meisler, M.H. Testosterone effects of kidney and urinary glycolipids in the light eared mouse mutant. FASEB Proc., 1979, 38, 405. Nanaka, G.; Kishimoto, Y. Simultaneous determination of picomole levels of gluco- and galactocerebroside, monogalactosyl diglyceride and sulfatides by high performance liquid chromatography. Biochim. Biophys. Acta, 1979, 572, 423. Suzuki, A.; Handa, S,; Yamakawa, T. Separation of molecular species of higher glycolipids by high performance liquid chromatography of their O-acetyl-N-p-nitrobenzoyl derivatives. J. Biochem., 1977, 82, 1185. Skipski, V.P. Thin-layer chromatography of neutral glycosphingolipids. Methods in Enzymology, 1975, 35, 396-425. Svennerholm, E.; Svennerholm, L. Isolation of blood serum glycolipids. Acta Chem. Scand., 1962, 16(5), 1282-1284. Gray, G.M. A comparison of the glycolipids found in different strains of Ascites tumour cells in mice. Nature, 1965, 207(4996), 505-507. Siddiqui, B.; McCluer, R.H. Lipid components of sialosylgalactosylceramide of human brain. J. Lipid Res., 1968, 9(3), 366-370. Puro, K. Isolation of bovine kidney gangliosides. Acta Chem. Scan., 1970, 24(1), 13-22. Siddiqui, B.; Hakomori, S. A ceramide tetrasaccharide of human erythrocyte membrane reacting of the anti-type. IV. pneumococcal polysaccharide antiserum. Biochim. Biophys. Acta., 1973, 330, 147-155. Yamakawa, I.; Irie, R.; Iwanaga, M. The chemistry of post-hemolytic residue of stroma of erythrocytes. IV. silicic acid chromatography of mammalian stroma glycolipids. J. Biochem. 1960, 48, 490-507.


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Liquid Chromatography

Radin, N.S. Florisil chromatography. Methods Enzymol., 1969, 14, 268-272. Rouser, G.; Bauman, A.J.; Kritchevsky, G.; Heller, D.; O'Brien, J. Quantitative chromatographic fractionation of complex lipid mixtures: brain lipids. J. Amer. Oil. Chem. Soc., 1961, 38, 544-555. Rouser, G.; Kritchevsky, G.; Heller, D.; Lieber, E. Lipid composition of beef brain, beef liver, and the sea anemone; two approaches to quantitative fractionation of complex lipid mixtures. J. Amer. Oil. Chem. Soc., 1963, 40, 425-554. Saito, T.; Hakomori, S. Quantitative isolation of total glycosphingolipids from animal cells. J. Lipid Res.,1971, 12(2) 257-259. Ando, S.; Isobe, M.; Nagai, M. High performance preparative column chromatography of lipids using a new porous silica, Iatrobeads . Biochim. Biophys. Acta, 1976, 424, 98-105. Yamakawa, T.; Yokoyama, S.; Kiso, N. Structure of main globoside of human erythrocytes. J. Biochem., 1962, 52, 228-231. Svennerholm, E.; Svennerholm, L. The separation of neutral blood-serum glycolipids by thin-layer chromatography. Biochim. Biophys. Acta., 1963, 70, 432-441. Martensson, E. Neutral glycolipids of human kidney. Isolation, identification, and fatty acid composition. Biochim. Biophys. Acta, 1966, 116, 296-308. Viswanathan, C.V.; Hayashi, A. Ascending dry-column chromatography as an aid in the preparative isolation of glycolipids. J. Chromat., 1976, 123, 243-246. Ullman, M.D.; McCluer, R.H. Isolation and quantitative analysis of neutral glycosylceramides by high performance liquid chromatography (HPLC). FASEB Proceedings, 1976. Tjaden, U.R.; Krol, J.H.; Van Hoeven, R.P.; Oomer-Meulemans, E.P.M.; Emmelot, P. High pressure liquid chromatography of glycosphingolipids (with special reference to gangliosides) J. Chromat.,1977, 136, 233-243. Folch, J . ; Lees, M.; Sloane-Stanley, G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem.,1957, 266, 497-509. (R)

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33.

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RECEIVED

December 10, 1979.


Cell Surface Glycolipids - American Chemical Society

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